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Transhydrogenase catalyses the transfer of reducing equivalents between NAD(H) and NADP(H) coupled to proton translocation across the membranes of bacteria and mitochondria. The protein has a tridomain structure. Domains I and III protrude from the membrane (e.g. on the cytoplasmic side in bacteria) and domain II spans the membrane. Domain I has the binding site for NAD ϩ /NADH, and domain III for NADP ϩ /NADPH. We have separately purified recombinant forms of domains I and III from Rhodospirillum rubrum transhydrogenase. When the two recombinant proteins were mixed with substrates in the stopped-flow spectrophotometer, there was a biphasic burst of hydride transfer from NADPH to the NAD ϩ analogue, acetylpyridine adenine dinucleotide (AcPdAD ϩ ). The burst, corresponding to a single turnover of domain III, precedes the onset of steady state, which is limited by very slow release of product NADP ϩ (kϷ0.03 s Ϫ1 ). Phase A of the burst (kϷ600 s Ϫ1 ) probably arises from fast hydride transfer in complexes of domains I and III. Phase B (kϷ10Ϫ50 s Ϫ1 ), which predominates when the concentration of domain I is less than that of domain III, probably results from dissociation of the domain I:III complexes and further association and turnover of domain I. Phases A and B were only weakly dependent on pH, and it is therefore unlikely that either the hydride transfer reaction, or conformational changes accompanying dissociation of the I:III complex, are directly coupled to proton binding or Keywords : transhydrogenase; stopped flow; proton translocation; recombinant protein; membrane protein.Transhydrogenase couples the transfer of reducing equivalents (hydride ion equivalents) between NAD(H) and NADP(H) to the translocation of protons across a membrane (for reviews,(1) The enzyme is found in animal mitochondria and in bacteria. Probably, under most physiological conditions, the reaction is driven to the right, towards NADPH formation, by the proton motive force generated by the respiratory (or photosynthetic) electron-transport chain.Transhydrogenase has three domains. Domains I and III protrude from the membrane and possess the nucleotide-binding sites ; domain I for NAD ϩ and NADH, and domain III for NADP ϩ and NADPH [1Ϫ3]. Domain II spans the membrane and might comprise 10Ϫ12 transmembrane helices [4].Recombinant forms of domain I from Rhodospirillum rubrum [5,6] and Escherichia coli [7,8] and purified. The isolated domains bind their respective nucleotides with high specificity and affinity [5Ϫ10, 11]. A mixture of the two domains from the R. rubrum protein catalyses transhydrogenation, even in the absence of the membrane-spanning domain II [6,9], but the rates of the 'forward' and 'reverse' transhydrogenation reactions catalysed by the mixture are very slow Ϫ they are profoundly limited by release of product NADPH (k off Ϸ 5ϫ10 Ϫ4 s Ϫ1 ) and NADP ϩ (k off Ϸ 0.03 s Ϫ1 ), respectively, from domain III [9]. Evidently, in the complete enzyme, release of NADP(H) is accelerated by domain II. The mixture of domains I and III...

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Transhydrogenase undergoes conformational changes to couple the redox reaction between NAD(H) and NADP(H) to proton translocation across a membrane. The protein comprises three components: dI, which binds NAD(H); dIII, which binds NADP(H); and dII, which spans the membrane. Experiments using isothermal titration calorimetry, analytical ultracentrifugation, and small angle x-ray scattering show that, as in the crystalline state, a mixture of recombinant dI and dIII from Rhodospirillum rubrum transhydrogenase readily forms a dI 2 dIII 1 heterotrimer in solution, but we could find no evidence for the formation of a dI 2 dIII 2 tetramer using these techniques. The asymmetry of the complex suggests that there is an alternation of conformations at the nucleotidebinding sites during proton translocation by the complete enzyme. The characteristics of nucleotide interaction with the isolated dI and dIII components and with the dI 2 dIII 1 heterotrimer were investigated. (a) The rate of release of NADP ؉ from dIII was decreased 5-fold when the component was incorporated into the heterotrimer. (b) The binding affinity of one of the two nucleotide-binding sites for NADH on the dI dimer was decreased about 17-fold in the dI 2 dIII 1 complex; the other binding site was unaffected. These observations lend strong support to the alternating-site mechanism.Transhydrogenase, found in the cytoplasmic membranes of bacteria, and in the inner membranes of animal mitochondria, couples the redox reaction between NAD(H) and NADP(H) to the translocation of protons.Its function in energy metabolism, biosynthesis, and detoxification has been discussed at length (1, 2). In different organisms (and possibly in different tissues of the same organism) transhydrogenase can either utilize the proton electrochemical gradient (⌬p) to drive reduction of NADP ϩ by NADH, or it can use NADPH oxidation by NAD ϩ to augment ⌬p formation. Energy coupling in transhydrogenase is indirect. In the forward direction (Reaction 1), protein conformational changes accompanying proton translocation bring together the nicotinamide rings of the bound nucleotides to allow the redox reaction (3). This "binding-change mechanism" may share common features with energy coupling in some ion-translocating ATPases. More generally, transhydrogenase has a number of properties that make it an excellent model for understanding the principles of operation of conformationally linked pumps in biology.The polypeptide organization of transhydrogenases varies between species, but the arrangement of the three components, dI, dII, and dIII, is essentially the same in all (Fig. 1). NAD(H) binds to dI, and NADP(H) binds to dIII; these two components protrude from the membrane (into the bacterial cytoplasm or mitochondrial matrix). The dII component spans the membrane, probably in 13 or 14 transmembrane helices (reviewed in Ref. 4). There is cross-linking and hydrodynamic evidence that both the bovine (5, 6) and Escherichia coli (7) transhydrogenases have two copies each of dI, dII, and dIII...

Transposon Tn917 mutagenesis of Bacillus subtilis BD99 followed by selection for protonophore resistance led to the isolation of strain MS119, which contained a single Tn917 insertion in an open reading frame whose deduced amino acid sequence was 56.6% identical to that of the Escherichia coli rho gene product. The insertional site was near the beginning of the open reading frame, which was located in a region of the B. subtilis chromosome near the spoOF gene; new sequence data for several open reading frames surrounding the putative rho gene are presented. The predicted B. subtilis Rho protein would have 427 amino acids and a molecular weight of 48,628. The growth of the mutant strain was less than that of the wild type on defined medium at 30°C. On yeast extract-supplemented medium, the growth of MS119 was comparable to that of the wild type on defined medium at 30°C. On yeast extract-supplemented medium, the growth of MS119 was comparable to that of the wild type at 30°C but was much slower at lower temperatures; sporulation occurred and competence was developed in cells of the mutant grown at 30°C. To determine whether the protonophore resistance and sensitivity to low growth temperature resulted from the insertion, a chloramphenicol resistance cassette was inserted into the wild-type B. subtilis rho gene of strain BD170; the resulting derivative displayed the same phenotype as MS119.The rho gene of Escherichia coli encodes an intensively studied transcriptional terminator that was first described by Roberts (29) and whose deduced amino acid sequence was determined by Pinkham and Platt (26). Rho protein is essential in E. coli; temperature-sensitive mutants have been isolated and are conditionally lethal (8). In Bacillus subtilis, the best-characterized gram-positive prokaryote at the molecular level, the status of a possible rho gene and protein product has been far more ambiguous. In 1980, Hwang and Doi (18) reported the isolation of a protein from B. subtilis that had similar properties to the E. coli Rho protein, i.e., poly(C)-dependent ATPase activity (albeit only 0.3% of that found in E. coli) and depression of transcription from +29 phage DNA by purified B. subtilis RNA polymerase. However, a subsequent examination of B. subtilis extracts revealed negligible RNA-dependent ATPase activity, and Western blotting (immunoblotting) with an anti-E. coli Rho antibody was also negative (4). Moreover, studies of the polar effects of mutations in B. subtilis trp and ilv genes indicated that such effects, although demonstrable in certain cases, were much less pronounced than in E. coli, in which the involvement of Rho is proposed (20,34,38). Thus, we were very interested in finding a B. subtilis gene with strong sequence similarity to the E. coli rho gene and whose deduced product would be comparable in size to the protein described by Hwang and Doi (18).The current finding emerged from a screening of Tn917 transposition libraries of B. subtilis for insertional mutations resulting in resistance to the protonoph...